Chir99021

WT and HGPS iPSCs were grown in colonies on mouse embryonic fibroblasts (MEF), inactivated with 10 mg/ml mitomycin C seeded at 30,000 cells/cm², and grown as previously described. For differentiation, embryonic bodies (EBs) are formed from hESC and iPSC clumps and grown on low attachment dishes in neural medium composed of 1/2 neurobasal and 1/2 HAM:F12 complemented with 2% of B-27 without VitA (Invitrogen) and 1% N-2 (Invitrogen). Induction of neural-crest differentiation was realized using CHIR-99021 (TOCRIS), LDN-193189 (Miltenyi) and SB 431542 (TOCRIS). Melanogenic commitment was realized with CHIR99021(TOCRIS), EDN3 (American peptide), SCF (peprotech), BMP-4 (peprotech) and ascorbic acid (Sigma-Aldrich). At day 7, EBs were plated on gelatine 0.1% and grown in MGM-4 medium supplemented with the same cytokines until around day 30.

The culture regimens to drive myogenic differentiation of hTERT-iMyoD-NHDFs or iMyoD-NHDFs in 2D cultures are illustrated in Fig. 2A. Doxycycline (Sigma-Aldrich) was added in culture media at 3 µg/ml throughout the whole culture period in each regimen. This concentration was chosen because the dose–response relationship between the Dox concentration and myogenic differentiation was examined in a previous study, and 3 µg/ml was determined to be the optimal concentration^{17 (link)}. Cells were seeded at 20% confluency and cultured in the NHDF medium for 7 days, trypsinized, re-plated at a density of 6 × 10⁴ cells/cm², cultured in the NHDF medium for 3 days, and then cultured in the KOSR medium (Knockout DMEM (Invitrogen) supplemented with 20% knockout serum replacement (Invitrogen), 1% MEM non-essential amino acids solution (Invitrogen), 1% glutamax (Invitrogen), and 50 U/ml penicillin/streptomycin (Invitrogen))^{40 (link)} for 4 days. The differences between the culture regimens are the supplements (5 µM CHIR99021 (Sigma-Aldrich) and/or 10 μM DAPT (Tocris)) in the NHDF and KOSR media after cell re-plating: no supplement for the DOX condition; CHIR99021 for the DOX + CHIR condition; DAPT for the DOX + DAPT condition; both CHIR99021 and DAPT for the DOX + CHIR + DAPT condition. All the media contained 100 µg/ml G418 and 100 ng/ml puromycin.

Human intestinal organoids (HIOs) were generated from iPSCs as previously described [15 (link)]. Briefly, iPSCs were passaged at approximately 80% confluency using a StemPro EZPassage Tool into 24-well tissue culture plates. Two days after passaging, mTeSR1 media were replaced with RPMI 1640 containing Activin A (100 ng/mL) and Wnt3A (25 ng/mL). On day 2 of differentiation, Wnt3A was removed and 0.2% and 2% FBS was added on days 2 and 3, respectively. Media were then replaced daily for 4 days with Advanced DMEM/F12 (Gibco) containing FBS (2%, v/v), FGF-4 (500 ng/mL), and CHIR99021 (3 µM, Tocris Bioscience, Minneapolis, MN, USA). Following differentiation, free floating and loosely attached intestinal tissue was embedded in Matrigel (Corning) and overlaid with Advanced DMEM/F12, B27 (1X), EGF (100 ng/mL), Noggin (100 ng/mL), CHIR99021 (2 µM, Tocris Bioscience), L-glutamine (5% v/v), and penicillin-streptomycin. All growth factors were from R&D systems unless otherwise noted.

Amicqui-1 were cultured on iMEF for at least 10 passages under two naïve conditions reported by Duggal et al., 2015 (link) and Zimmerlin et al., 2016 (link). Duggal medium is composed of hESC medium (DMEM/F-12, 20% KOSR, non-essential amino acids and mercaptoethanol) supplemented with 10 ng/mL FGF2, 1000 U LIF (Peprotech, 300–05), 1 µM PD0325901 (Tocris, 4192), 3 µM CHIR99021 (Tocris, 4423), 10 µM forskolin (Stemgent, 04–0025), and 50 ng/mL acid ascorbic (Sigma-Aldrich, A4544). Zimmerlin medium is constituted by hESC medium supplemented with 1000 U LIF, 1 µM PD0325901, 3 µM CHIR99021, and 4 µM XAV939 (Tocris, 3748). The passages were carried out every 3–5 days using Accutase. Cells in passage 7 were used for detection of naïve pluripotency markers by qPCR.

CAG33 human iPSC (hiPSCs) (ND36997) and CAG180 iPSCs (ND36999) harbouring HTT alleles with 33 and 180 CAG repeats, respectively, were obtained from the NINDS iPSC Repository at Coriell Institute. The expanded HTT allele in the CAG180 hiPSC line was corrected to 18 CAG repeats by homologous recombination to generate isogenic control hiPSC lines: isoHD-Corr-1, isoHD-Corr-2, and isoHD-Corr-3^{42 (link)}. All hiPSCs were cultured on Matrigel-coated plates in mTeSR-1 medium (STEMCELL Technologies, #05850) in a cell culture incubator at 37 °C and 5% CO₂.
hiPSCs were induced into pNSCs according to a previously published protocol^{61 (link)}. Briefly, hiPSCs at about 20% confluence were treated with N2B27 media (DMEM-F12/Neural Basal medium 1:1 with 1% N2, 2% B27, 1% pen/strep/glutamine, 10 ng ml⁻¹ hLIF, and 5 μg ml⁻¹ BSA) containing 3 μM CHIR99021 (Tocris), 2 μM SB431542 (Sigma) and 0.1 μM compound E (Millipore) for the first 7 days. The culture was then split 1:3 for the next six passages using Accutase without compound E on Matrigel-coated plates. Cells at passage 5 were used for experiments.